Skip to main content
PMC home page
Biochemical Journal logoLink to Biochemical Journal
. 2001 Oct 15;359(Pt 2):255–263. doi: 10.1042/0264-6021:3590255

Coactosin-like protein, a human F-actin-binding protein: critical role of lysine-75.

P Provost 1, J Doucet 1, A Stock 1, G Gerisch 1, B Samuelsson 1, O Rådmark 1
PMCID: PMC1222143  PMID: 11583571

Abstract

Coactosin-like protein (CLP) was recently identified in a yeast two-hybrid screen using 5-lipoxygenase as bait. In the present study, we report the functional characterization of CLP as a human filamentous actin (F-actin)-binding protein. CLP mRNA shows a wide tissue distribution and is predominantly expressed in placenta, lung, kidney and peripheral-blood leucocytes. Endogenous CLP is localized in the cytosol of myeloid cells. Using a two-hybrid approach, actin was identified as a CLP-interacting protein. Binding experiments indicated that CLP associates with F-actin, but does not form a stable complex with globular actin. In transfected mammalian cells, CLP co-localized with actin stress fibres. CLP bound to actin filaments with a stoichiometry of 1:2 (CLP: actin subunits), but could be cross-linked to only one subunit of actin. Site-directed mutagenesis revealed the involvement of Lys(75) of CLP in actin binding, a residue highly conserved in related proteins and supposed to be exposed on the surface of the CLP protein. Our results identify CLP as a new human protein that binds F-actin in vitro and in vivo, and indicate that Lys(75) is essential for this interaction.

Full Text

The Full Text of this article is available as a PDF (368.7 KB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Andrade M. A., Chacón P., Merelo J. J., Morán F. Evaluation of secondary structure of proteins from UV circular dichroism spectra using an unsupervised learning neural network. Protein Eng. 1993 Jun;6(4):383–390. doi: 10.1093/protein/6.4.383. [DOI] [PubMed] [Google Scholar]
  2. Blomster M., Wetterholm A., Mueller M. J., Haeggström J. Z. Evidence for a catalytic role of tyrosine 383 in the peptidase reaction of leukotriene A4 hydrolase. Eur J Biochem. 1995 Aug 1;231(3):528–534. doi: 10.1111/j.1432-1033.1995.0528d.x. [DOI] [PubMed] [Google Scholar]
  3. Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1006/abio.1976.9999. [DOI] [PubMed] [Google Scholar]
  4. Chen K. S., Manian P., Koeuth T., Potocki L., Zhao Q., Chinault A. C., Lee C. C., Lupski J. R. Homologous recombination of a flanking repeat gene cluster is a mechanism for a common contiguous gene deletion syndrome. Nat Genet. 1997 Oct;17(2):154–163. doi: 10.1038/ng1097-154. [DOI] [PubMed] [Google Scholar]
  5. Corpet F., Servant F., Gouzy J., Kahn D. ProDom and ProDom-CG: tools for protein domain analysis and whole genome comparisons. Nucleic Acids Res. 2000 Jan 1;28(1):267–269. doi: 10.1093/nar/28.1.267. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Drubin D. G., Mulholland J., Zhu Z. M., Botstein D. Homology of a yeast actin-binding protein to signal transduction proteins and myosin-I. Nature. 1990 Jan 18;343(6255):288–290. doi: 10.1038/343288a0. [DOI] [PubMed] [Google Scholar]
  7. Faix J., Steinmetz M., Boves H., Kammerer R. A., Lottspeich F., Mintert U., Murphy J., Stock A., Aebi U., Gerisch G. Cortexillins, major determinants of cell shape and size, are actin-bundling proteins with a parallel coiled-coil tail. Cell. 1996 Aug 23;86(4):631–642. doi: 10.1016/s0092-8674(00)80136-1. [DOI] [PubMed] [Google Scholar]
  8. Friederich E., Vancompernolle K., Huet C., Goethals M., Finidori J., Vandekerckhove J., Louvard D. An actin-binding site containing a conserved motif of charged amino acid residues is essential for the morphogenic effect of villin. Cell. 1992 Jul 10;70(1):81–92. doi: 10.1016/0092-8674(92)90535-k. [DOI] [PubMed] [Google Scholar]
  9. Fulgenzi G., Graciotti L., Granata A. L., Corsi A., Fucini P., Noegel A. A., Kent H. M., Stewart M. Location of the binding site of the mannose-specific lectin comitin on F-actin. J Mol Biol. 1998 Dec 18;284(5):1255–1263. doi: 10.1006/jmbi.1998.2294. [DOI] [PubMed] [Google Scholar]
  10. Heintz D., Kany H., Kalbitzer H. R. Mobility of the N-terminal segment of rabbit skeletal muscle F-actin detected by 1H and 19F nuclear magnetic resonance spectroscopy. Biochemistry. 1996 Oct 1;35(39):12686–12693. doi: 10.1021/bi961159k. [DOI] [PubMed] [Google Scholar]
  11. Ishikawa R., Hayashi K., Shirao T., Xue Y., Takagi T., Sasaki Y., Kohama K. Drebrin, a development-associated brain protein from rat embryo, causes the dissociation of tropomyosin from actin filaments. J Biol Chem. 1994 Nov 25;269(47):29928–29933. [PubMed] [Google Scholar]
  12. Kozak M. An analysis of 5'-noncoding sequences from 699 vertebrate messenger RNAs. Nucleic Acids Res. 1987 Oct 26;15(20):8125–8148. doi: 10.1093/nar/15.20.8125. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Lappalainen P., Kessels M. M., Cope M. J., Drubin D. G. The ADF homology (ADF-H) domain: a highly exploited actin-binding module. Mol Biol Cell. 1998 Aug;9(8):1951–1959. doi: 10.1091/mbc.9.8.1951. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Marriott G., Zechel K., Jovin T. M. Spectroscopic and functional characterization of an environmentally sensitive fluorescent actin conjugate. Biochemistry. 1988 Aug 23;27(17):6214–6220. doi: 10.1021/bi00417a004. [DOI] [PubMed] [Google Scholar]
  15. McGough A. F-actin-binding proteins. Curr Opin Struct Biol. 1998 Apr;8(2):166–176. doi: 10.1016/s0959-440x(98)80034-1. [DOI] [PubMed] [Google Scholar]
  16. Pouliot M., McDonald P. P., Krump E., Mancini J. A., McColl S. R., Weech P. K., Borgeat P. Colocalization of cytosolic phospholipase A2, 5-lipoxygenase, and 5-lipoxygenase-activating protein at the nuclear membrane of A23187-stimulated human neutrophils. Eur J Biochem. 1996 May 15;238(1):250–258. doi: 10.1111/j.1432-1033.1996.0250q.x. [DOI] [PubMed] [Google Scholar]
  17. Prendergast F. G., Meyer M., Carlson G. L., Iida S., Potter J. D. Synthesis, spectral properties, and use of 6-acryloyl-2-dimethylaminonaphthalene (Acrylodan). A thiol-selective, polarity-sensitive fluorescent probe. J Biol Chem. 1983 Jun 25;258(12):7541–7544. [PubMed] [Google Scholar]
  18. Provost P., Doucet J., Hammarberg T., Gerisch G., Samuelsson B., Radmark O. 5-Lipoxygenase interacts with coactosin-like protein. J Biol Chem. 2001 Jan 31;276(19):16520–16527. doi: 10.1074/jbc.M011205200. [DOI] [PubMed] [Google Scholar]
  19. Provost P., Samuelsson B., Rådmark O. Interaction of 5-lipoxygenase with cellular proteins. Proc Natl Acad Sci U S A. 1999 Mar 2;96(5):1881–1885. doi: 10.1073/pnas.96.5.1881. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Rees M. K., Young M. Studies on the isolation and molecular properties of homogeneous globular actin. Evidence for a single polypeptide chain structure. J Biol Chem. 1967 Oct 10;242(19):4449–4458. [PubMed] [Google Scholar]
  21. Röhrig U., Gerisch G., Morozova L., Schleicher M., Wegner A. Coactosin interferes with the capping of actin filaments. FEBS Lett. 1995 Oct 30;374(2):284–286. doi: 10.1016/0014-5793(95)01130-7. [DOI] [PubMed] [Google Scholar]
  22. Safer D. An electrophoretic procedure for detecting proteins that bind actin monomers. Anal Biochem. 1989 Apr;178(1):32–37. doi: 10.1016/0003-2697(89)90351-5. [DOI] [PubMed] [Google Scholar]
  23. Samuelsson B. Leukotrienes: mediators of immediate hypersensitivity reactions and inflammation. Science. 1983 May 6;220(4597):568–575. doi: 10.1126/science.6301011. [DOI] [PubMed] [Google Scholar]
  24. Scheel J., Ziegelbauer K., Kupke T., Humbel B. M., Noegel A. A., Gerisch G., Schleicher M. Hisactophilin, a histidine-rich actin-binding protein from Dictyostelium discoideum. J Biol Chem. 1989 Feb 15;264(5):2832–2839. [PubMed] [Google Scholar]
  25. Vaduva G., Martin N. C., Hopper A. K. Actin-binding verprolin is a polarity development protein required for the morphogenesis and function of the yeast actin cytoskeleton. J Cell Biol. 1997 Dec 29;139(7):1821–1833. doi: 10.1083/jcb.139.7.1821. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Van Troys M., Dewitte D., Goethals M., Carlier M. F., Vandekerckhove J., Ampe C. The actin binding site of thymosin beta 4 mapped by mutational analysis. EMBO J. 1996 Jan 15;15(2):201–210. [PMC free article] [PubMed] [Google Scholar]
  27. Vancompernolle K., Vandekerckhove J., Bubb M. R., Korn E. D. The interfaces of actin and Acanthamoeba actobindin. Identification of a new actin-binding motif. J Biol Chem. 1991 Aug 15;266(23):15427–15431. [PubMed] [Google Scholar]
  28. Vandekerckhove J. Actin-binding proteins. Curr Opin Cell Biol. 1990 Feb;2(1):41–50. doi: 10.1016/s0955-0674(05)80029-8. [DOI] [PubMed] [Google Scholar]
  29. Vandekerckhove J., Van Damme J., Vancompernolle K., Bubb M. R., Lambooy P. K., Korn E. D. The covalent structure of Acanthamoeba actobindin. J Biol Chem. 1990 Aug 5;265(22):12801–12805. [PubMed] [Google Scholar]
  30. Whalen R. G., Butler-Browne G. S., Gros F. Protein synthesis and actin heterogeneity in calf muscle cells in culture. Proc Natl Acad Sci U S A. 1976 Jun;73(6):2018–2022. doi: 10.1073/pnas.73.6.2018. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. de Hostos E. L., Bradtke B., Lottspeich F., Gerisch G. Coactosin, a 17 kDa F-actin binding protein from Dictyostelium discoideum. Cell Motil Cytoskeleton. 1993;26(3):181–191. doi: 10.1002/cm.970260302. [DOI] [PubMed] [Google Scholar]

Articles from Biochemical Journal are provided here courtesy of The Biochemical Society

RESOURCES